Inherited disorders of production of the β-chain of adult hemoglobin A (β-thalassemia) or mutations affecting the structure of the β-globin chain (sickle cell disease) are the most common genetic diseases in the world, afflicting millions of individuals worldwide, and are designated by WHO as a global health burden. Pharmacological augmentation of fetal hemoglobin (γ-globin chain) production, to replace the defective or missing β-globin chains, has emerged as a promising therapeutic modality.
Anemia, a red blood cell disorder, can be grossly defined as a reduction in the ability of blood to transport oxygen. Although red blood cell disorders may be caused by certain drugs and immune system disorders, the majority are caused by genetic defects in the expression of hemoglobin. Disorders of hemoglobin synthesis include deficiencies of globin synthesis such as thalassemia syndromes and structural abnormalities of globin such as sickle cell syndromes and syndromes associated with unstable hemoglobins.
Fetal globin (also known as gamma globin or y globin) normally combines with alpha globin chains prenatally to form fetal hemoglobin (HbF). Fetal globin is replaced by beta globin after birth, which then combines with alpha globin to form adult hemoglobin A. Fetal globin performs the same function as beta globin, and can combine with the alpha chains to generate a healthy form of hemoglobin thereby reducing high concentrations of unmatched alpha globin chains.
The various types of β thalassemias are syndromes resulting from mutations which produce a deficiency of β globin chains. In beta thalassemia, the unmatched alpha globin chains aggregate inside red blood cells (RBCs) and their progenitors, causing the premature destruction of RBCs and RBC progenitors, which results in anemia, transfusion-dependence, iron overload, organ failure, and early death.
In sickle cell disease (SCD), one amino acid substitution in the beta globin chain results in the generation of sickling hemoglobin (HbS), which allows polymerization with repeated cycles of deoxygenation. Polymerization results in “sickling” of RBCs. The sickled RBCs undergo hemolysis, while adhesive sickled RBCs occlude the microcirculation, provoking widespread tissue ischemia and organ infarction. The natural history of SCD is marked by painful crises and acute chest syndrome and eventual potentially life-threatening sequelae, including renal insufficiency, retinitis, osteonecrosis, osteomyelitis, aplastic crises, functional asplenism, stroke, priapism, and severe pulmonary hypertension.
Many efforts to stimulate HbF production have accordingly been undertaken, but pharmacologic reactivation of high-level HbF expression with non-toxic and tolerable therapeutic agents that are orally-available (for worldwide therapeutic application) has been an elusive therapeutic goal for many years. In sickle cell disease, average HbF levels in adult patients are 5-7%; but levels of HbF >15-20% and >70% F-cells are typically required to ameliorate most of the clinical complications. One HbF stimulant therapeutic, hydroxyurea (HU), is FDA-approved for treatment of sickle cell disease and benefits approximately 40% of subjects, with most benefit occurring those who attain absolute HbF levels >0.5 g/dl or 20%. HbF levels achieved are often not sufficiently high to completely ameliorate all complications. Additional therapeutics, especially non-cytotoxic agents which can be used in combinations with HU, could provide additional benefit. There are no therapeutic agents approved for the β-thalassemia syndromes.
Individuals with severe sickle cell anemia develop no symptoms until about five to six months after birth. In these infants it was determined that fetal hemoglobin did not interact with HbS and, as long as sufficient quantities were present, could modulate the effects of HbS disease. This modulating effect of β globin is also observed with other β globin disorders, such as HbC and HbD, and other mutations of the β chain. HbS polymerization is also significantly affected by the hemoglobin concentration of the cell. The higher the HbS concentration, the greater the chances for contact between two or more HbS molecules. Dehydration increases hemoglobin concentration and greatly facilitates sickling.
The thalassemia syndromes are a heterogeneous group of disorders all characterized by a lack of or a decreased synthesis of the globin chains of HbA. Deficiencies of β-globin expression are referred to as β-thalassemias and deficiencies of α-globin, α-thalassemias. The hemolytic consequences of deficient globin chain synthesis result from decreased synthesis of one chain and also an excess of the complementary chain. Free chains tend to aggregate into insoluble inclusions within erythrocytes causing premature destruction of maturing erythrocytes and their precursors, ineffective erythropoiesis, and the hemolysis of mature red blood cells. The underlying defects of hemoglobin synthesis have been elucidated over the years and largely reside in the nucleic acid sequences which express or control the expression of a or p globin protein.
Beta thalassemias are genetic disorders of moderate to severe anemias, caused by molecular mutations which decrease production of the beta globin chain of adult hemoglobin A (α2β2). Unmatched alpha globin is toxic and causes early apoptosis of red blood cell precursors. In thalassemia major, patients have severe anemia and require red blood cell transfusions regularly to survive, beginning in infancy. In thalassemia intermedia, there is moderate, chronic hemolytic anemia, which adversely affects growth, cardiac function, and other systems; transfusions are used intermittently in childhood but often required regularly later in life. Complications of blood transfusions, iron overload and hepatitis C, cause widespread organ damage and early mortality. Beta hemoglobinopathies, or sickle cell syndromes, caused by a single point mutation (A-T) in the beta globin gene, causes polymerization of HbS, distortion of the red blood cell, chronic hemolysis, vascular adhesion, tissue hypoxia, and widespread organ damage. Fetal hemoglobin, (HbF, α2γ2), is an alternative type of hemoglobin that is normally silenced in early childhood. Renewed or increased fetal globin (HbF) expression replaces the missing adult globin protein and reduces anemia in beta thalassemia, and inhibits sickling in sickle cell disease, preventing almost all the complications of this disease, when present in adequate amounts.
In the US and EU, beta thalassemias are niche orphan conditions, with an estimated 1200 patients in the US and 6500 patients in the EU, primarily in Italy, Greece, and the UK, with small populations in Germany and France. In the US, sickle cell disease is also an orphan condition with 80-100,000 patients. Ex-US, these conditions are considered a major global health burden, with 500,000 patients estimated in SE Asia.
A small number of therapeutic agents of different chemical classes can induce HbF experimentally, with only a few are orally-active or currently in clinical testing. Three general classes of therapeutic agents have been shown to induce HbF significantly in subjects with sickle cell disease and β-thalassemia, including: cytotoxic chemotherapeutic agents (such as Hydroxyurea (HU), 5-azacytidine, and decitabine), erythropoietin (EPO) preparations, and short chain fatty acids (SCFAs) and derivatives (SCFADs) which include some HDAC inhibitors. Additionally, there are a variety of small molecules have been shown to effect hemoglobin or fetal globin expression. Early experiments demonstrated that acetate (CH3COOH), propionate (CH3CH2COOH), butyrate (CH3CH2CH2COOH) and isobutyrate (CH3CH(CH3)COOH) all induced hemoglobin synthesis in cultured Friend leukemia cells (E. Takahashi et al., Gann 66:577-80, 1977). Additional studies showed that polar compounds, such as acid amides, and fatty acids could stimulate the expression of both fetal and adult globin genes in murine erythroleukemia cells (U. Nudel et al., Proc. Natl. Acad. Sci. USA 74:1100-4, 1977). Hydroxyurea (H2NCONHOH), another relatively small molecule, was found to stimulate globin expression (N. L. Letvin et al., N. Engl. J. Med. 310:869-73, 1984). Stimulation, however, did not appear to be very specific to fetal globin (S. Charache et al., Blood 69:109-16, 1987). Hydroxyurea (HU) is also a well-known carcinogen making its widespread and long term use as a pharmaceutical impractical. One of the major breakthroughs in the treatment of hemoglobinopathies was made when it was discovered that butyric acid (butanoic acid; CH3CH2CH2COOH) accurately and specifically stimulated transcription of the human fetal globin (HbF or γ-globin) gene (G. A. Partington et al., EMBO J. 3:2787-92, 1984). Some of these have shown proof-of-principle, but, except for HU, have required parenteral administration or large doses, which were not suitable for broad application.
While three short chain fatty acid (SCFA) agents have been reported to induce γ-globin expression and to increase hemoglobin levels in subjects with β-thalassemia, rendering some β-thalassemia subjects transfusion-independent, these prior generations of SCFAs, including arginine butyrate (AB) and sodium phenylbutyrate (SPB), have limited utility as a therapeutic agent in vivo, as they are either rapidly metabolized, required intravenous (IV) infusions, or required large doses which were difficult for subjects to tolerate long-term. Furthermore, these 1st generation SCFAs are also known to inhibit erythroid cell proliferation, and therefore require titration and intermittent dosing, complicating their use in conditions of anemia where compensatory erythroid cell proliferation is desirable. There, thus remains an unmet clinical need for a therapeutic agent that induces γ-globin gene expression and does not inhibit erythroid cell proliferation (i.e., is not cytotoxic), and which is more applicable for wide application in these genetic diseases.
Despite long-term efforts, regulatory approval has been obtained for only one chemotherapeutic agent. Pharmacologic reactivation of high-level HbF expression with non-cytotoxic, tolerable therapeutics is still an unmet medical need for this global health burden. Arginine Butyrate and Phenylbutyrate have demonstrated proof-of-principle in re-inducing expression of HbF, correcting globin imbalance, increasing hemoglobin levels, and reducing transfusion requirements, these HDAC inhibitors, however, were inconvenient for broad application due to high dose requirements and/or IV administration. Accordingly, there is an unmet need for low dosing, long-term use of orally available and high activity agents which induce HbF expression which are safe for use in a large patient base.